Purification and characterization of MAR1 -: A mitochondrial associated ribonuclease from Leishmania tarentolae
Purification and characterization of MAR1 -: A mitochondrial associated ribonuclease from Leishmania tarentolae
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DOI：
10.1074/jbc.273.45.30003
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发表时间：
1998-11-06
影响因子：
4.8
通讯作者：
Simpson, L
中科院分区：
文献类型：
--
作者：
Alfonzo, JD;Thiemann, OH;Simpson, L
一个相对耐热的22 kda核糖核酸内切酶（MAR1）从利什曼原虫的线粒体提取物中纯化了超过10,000倍，并关闭了该基因。根据RNA底物的不同，纯化的核酸酶的K-m值为100-145 +/- 33 nM， V-max值为1.8-2.9 +/- 2 nmol/min，并产生3'- oh和5'-磷酸。切割仅限于在所测试的底物RNA中的几个特定位点，但预编辑RNA的切割通常不依赖于同源引导RNA的添加。MAR1基因在大肠杆菌和荚膜乳杆菌细胞中表达，重组蛋白经亲和纯化。该重组酶的裂解特异性与原酶相同。单拷贝MAR1基因映射到一条820千碱基对的染色体上，包含579个核苷酸的开放阅读框。18个氨基酸组成的n端序列显示出线粒体未裂靶序列的特征。在数据库检索中发现了两个MAR1同源基因，分别对应于隐杆线虫（Caenorhabditis elegans） （GenBank（TM）登录号Z69637）和fulgidus (Archaeoglobus fulgidus) （GenBank（TM）登录号AE000943）的未知开放阅读框。MAR1在l.t arentolae线粒体RNA代谢中的功能尚不清楚。
A relatively thermostable 22-kDa endoribonuclease (MAR1) was purified more than 10,000-fold from a mitochondrial extract of Leishmania tarentolae and the gene closed. The purified nuclease has a K-m of 100-145 +/- 33 nM and a V-max of 1.8-2.9 +/- 2 nmol/min, depending on the RNA substrate, and yields a 3'-OH and a 5'-phosphate. Cleavage was limited to several specific sites in the substrate RNAs tested, but cleavage of pre-edited RNAs was generally independent of the addition of cognate guide RNA. The MAR1 gene was expressed in Escherichia coli or in L. tarentolae cells, and the recombinant protein was affinity-purified. The cleavage specificity of the recombinant enzyme from L. tarentolae was identical to that of the native enzyme. The single copy MAR1 gene maps to an 820-kilobase pair chromosome and contains an open reading frame of 579 nucleotides. The 18-amino acid N-terminal sequence shows characteristics of an uncleaved mitochondrial targeting sequence. Data base searching revealed two homologues of MAR1 corresponding to unidentified open reading frames in Caenorhabditis elegans (GenBank(TM) accession number Z69637) and Archaeoglobus fulgidus (GenBank(TM) accession number AE000943). The function of MAR1 in mitochondrial RNA metabolism in L. tarentolae remains to be determined.